Document MG9z6dMy5bB75488pB7GDpmYy

ire of litical must >vernth as tilted i-[he ns of ts the , ained e--is initia ls the i l'ott, ithani ingly eases I ecoII y of elivi.xt ctivist C. T. iicago, seeve/ e its Burt, Iculus. . icfcen. trvaril PREDICTIVE VALUE OF CARCINOGENESIS BIOASSAYS Cesare Maltoni Institute oj Oncology and Tumour Centre Bologna, Italy It has been estimated that from 80 to 90C< of human tumors are caused by factors present in the'occupational and general environment. Therefore, cancer must be largely considered an ecologic disease. On the other hand, the number and quantity of oncogenic agents in the occupational and general environment have progressively increased in the last few decades, due mainly to the following factors: the concentration of onco genic agents already present in the Earth's surface, surfacing of oncogenic agents from beneath the Earth's surface, and production of new potentially oncogenic compounds by chemical and petrochemical industries. The major problem is now represented by the multitude of products of synthetic chemical industries, especially the petrochemical industry, that are unknown to animal and human protoplasm and of whose effects we are there fore fully ignorant. Factories represent the observatory: there, the compounds are planned, produced, and released into the general environment as consumer goods and pollution; there, the most exposed population works. In this situation, one should be therefore aware that tools arc needed for assessing the risk of agents concentrated, or brought, or introduced de novo into the occupational and general human environment. This need is strengthened by the following basic facts: The potentially oncogenic agents in the human environment are progres sively increasing. Different oncogenic agents may have additive effects. Changes produced by oncogenic agents are largely irreversible. Oncogenic agents may exert their effects on different organs and tissues and widely affect the target organs. A large part of the natural history of tumors occurs without any clinically, and sometimes otherwise, detectable pathologic changes. Cancer is not a reversible disease. The potential oncogenic risk for man by occupational and environmental agents has so far been identified when the incidence of a particular type of tumor in an exposed population has been especially high or on the basis of retrospective epidemiologic studies. The gravity of the present situation, however, no longer permits the old policy of "let's wait and see." In other words, we must now predict oncogenic risks to avoid human exposure to such agents. It is a fact that the three most important cases of environmental and occupational tumors, discovered after 1970, were indirectly or directly predicted experimentally. The first case was the adolescent clear-cell vaginal adenocarcinoma found in girls born from mothers treated during pregnancy with synthetic nonstcroid 432 Annals New York Academy of Sciences estrogen therapy. It should be noted that, in 1938, Lacassagne 1 had already reported that mammary carcinomas arose in male mice treated with stilbestrol; several years later, it was shown, by several scientists, that the same hormone was producing a variety of tumors in hormone-dependent and -independent tissues among different experimental animal species.2 The second case was pulmonary carcinoma among workers exposed to bis(chloromcthyl) ether. This new type of occupational tumor was discovered by experimental evidence: Van Duuren et al* and Laskin et al:' showed, re spectively, that, when injected subcutaneously into rats or applied to the skin of mice, the compound was producing subcutaneous fibrosarcomas and skin carcinomas, and that, when inhaled by rats, it induced squamous cell carcinomas of the lung. The third example is the history of vinyl chloride cancerogenicity.5-10 In 1971, following our early observations on the increased incidence of atypical cells in the sputa of workers exposed to vinyl chloride and the results of Viola et at12 which showed that rats that experimentally inhaled 30,000 ppm of this monomer developed Zymbal gland carcinomas, we undertook a large program of experiments to study the effects of vinyl chloride, administered at different doses and by different routes, on animals of different species, strains, sexes, and ages. In August 1972, we realized that this compound was producing angio sarcomas of the liver and of other sites and nephroblastomas other than Zymbal gland carcinomas. These results were communicated by us, among others, to the factories that supported our studies, and to the Manufacturing Chemists Association, to enable them to adopt adequate preventive measures and to promote these epidemiologic investigations, which in December 1973 first identified an occupational tumor, a liver angiosarcoma in a worker of a United States factory (B. F, Goodrich in Louisville, Kentucky) that was producing vinyl chloride-polyvinyl chloride fVC-PVC). No histologic difference could be found among the angiosarcomas in animals and in man exposed to vinyl chloride. Updated results of some experiments on VC carcinogenicity are shown in Table 1-9. The onset of several hepatomas among animals experimentally treated with VC (Table 10) is particularly relevant, in light of recent observations by Selikoff" and Popper" of a few cases of these tumors among workers of VC-PVC industries. . All types of tumors in man that have so far. been found to be correlated in some way to VC exposure are included among those already observed in animals (Table 11).' Experiments are now underway in our laboratories on the effects on rats of inhalational exposure to 25, 10. 5, and I ppm of VC and of oral administra tion of 1, 0.3, and 0.03 mg/kg of body weight of the monomer, with the same schedule of experiments BTI and BT11, respectively. From the results presented on VC carcinogenicity in laboratory animals, it appears that experimental bioassays may predict carcinogenicity, give indica tions of target organs, give information on the risk level based on the doseresponse relationship, and may be able to assess the risk in function of the route of exposure. At present, much emphasis is focused on VC carcinogenicity. We hope that CJ c/i 3 Cv 3 o &: 3 o 3 ~ n r,, cfr -o OC OO <--s o-- y j r*t-- 23! 23". 3v 3c. -o ?n S3 noa Table 1 Experiments BTI and BT6: ... .. -- Exposure bv Inhalation to VC in Air at 30.000, 10,000, 6000, 2500, 500, 250 and 50 ppm for 4 hr. Daily, 5 Days per Week for 52 Weeks. Results After a 135-WEEK-Eeriod -rrrtT'r' - - Groups and Treatment Total No. of Animals (SpragueDawley rats) $ and cf Zymbal Gland Carci nomas I VC, 30,000 ppm II VC, 10,000 ppm III VC, 6000 ppm IV VC, 2500 ppm V VC, 500 ppm VI VC, 250 ppm VII VC, 50 ppm VIII No treatment Total 60 69 72 74 67 67 64 68 541 35 16 7 2 4 -- -- -- 64 Nephro blasto mas -- 5 4 6 4 6 I -- 26 No. of Animais with Tumors Angiosarcomas Other Liver Sites Subcu taneous Angio mas Skin Carci nomas * Hepa tomas 18 1 93 13 3 13 3 72 42 11 ---- 65 15 1I1 431 31I 312 1 I3 -- 4-- 1 1-- -- -- __ 13 12 8 Brain Neuro blasto mas Mam mary Carcino mas Pregastric Papillo- . mas 1 2 11 7 3-- 3---- 5 1-- -- 1-- -- 1-- -- 2-- __ __ __ 16 10 11 2_ oa *-toTiJ Q. o* t<o ' < n O CD 5' t: * Most of these tumors occurred in the sebaceous glands. u> i 434 Annals New York Academy of Sciences Table 2 Experiment BT2: Exposure by Inhalation to VC in Air at 200, 150, and 100 ppm for 4 Hr Daily, 5 Days per Week for 52 Weeks. Results After 89 Weeks Groups and Treatment I VC, 200 ppm II VC, 150 ppm III VC, 100 ppm IV No treatment Total Animals (Sprague-Dawley rats) $ and ________ ________ _ Total Survivors 120 41 120. 45 120 49 185 76 545 211 No. of Animals with Tumors Gland Carcino- Nephro- mas blastomas Angiosarcomas ---------------------- Other Liver Sites -- 2 71 ____ 4 3 1 1 8 1-- 1 -- --1 2 14 11 3 this emphasis will not mask the more general problems and the multitude of needs of occupational cancerogenesis. In our Institute, other compounds have been or are being tested. Experi mental bioassays have been performed on chromium compounds and other inorganic pigments by subcutaneous injection of rats (Table 12). These results point out the potential risk of some of the most widespread inorganic pigments. Experiments have also been performed to assess the oncogenic potential of asbestos on peritoneal mesothelium, by injecting crocidolite into the abdominal cavity (Table 13). Our data are consistent with the well-known epidemiologic results that point out the high risk among asbestos workers of developing peritoneal mesotheliomas. Carcinogenicity bioassays on several other compounds produced and dis tributed on a large scale are now underway; the compounds being tested include monomers used in plastic industries, such as styrene, acrylonitrile, and vinylidene chloride: polymers; estrogens and progestins; and petroleum proteins. Let us hope that experimental bioassays will no longer be procrastinated due to skepticism of their validity, based on past results; fatalistic resignation, because of the huge number of newly produced comp'ounds: and complications of elaborate testing procedures. In regard to skepticism, I think that we should now critically review the methods and results of past experiments, which may now appear to have been poorly conducted. With regard to the large number of compounds, it is true that there are already millions of newly produced chemicals; however, those that urgently need to be tested, mainly because of their widespread use, actually number only in the hundreds. Concerning complications in performing elaborate testing procedures, it is true that the more experimental tests on animals reproduce the conditions of human exposure, the more relevant they are to man; it should be pointed out, EC- 1935 3n> Cr>. tit ijz n O iCnJ *o 2 Table 3 Experiment BT3: Exposure by Inhalation to VC in Air at 10,000, 6000, 2500,500 250 and 50 ppm 4 Hr Daily, 5 Days per Week for 17 Weeks. Results After 114 Weeks* Groups and Treatment I VC, 10,000 ppm II VC, 6000 ppm 111 VC, 2500 ppm IV VC, 500 ppm V VC, 250 ppm VI VC, 50 ppm VII No treatment Total Animals (Sprague-Dawley) rats) ? and <f Total Survivors 60 1 60 3 60 3 60 10 60 4 60 9 190 35 550 65 Zymbal Gland Carcinomas 6 (16) 6 (7) 3 (2) 1 (4) -- -- -- 16 (29) No. of Animals with Tumors Nephrobla stomas Angiosarcomas Liver Other Sites 1 (5) I (4) 2 (6) -- (4) '> (6) -- -- 6 (25) -- (9) 1 (13) 1 (13) 1 (7) -- (4) -- -- 3 (46) 1 (3) 1 (3) 2 (3) 1 (2) -- (2) 2 1 8 (13) Brain Neurobla stomas 6 (7) 2 (3) 2 (5) -- -- -- -- 10 (15) 2 EL o 3 *0 OCL O <n> <3 a> o CO 53' in Vt>i Vi * In parentheses are recorded the number of tumors observed after 114 weeks among the Sprague-Dawley rats in experiment BTlt in which the animals were treated for 52 weeks. LAUn> i V -JOU safe 436 A nnals New Y o rk Academ y o f Sciences EC- 1936 Table 4 Experiment BT7: Exposure by Inhalation to VC in Air at 10,000, 6000, 2500, 500, 250 and 50 ppm *^or 4 Hr Daily, 5 Days per Week for 52 Weeks. Results After 88 Weeks * Groups ... and Treatment Animals (Wistar rats) Total Survivors No. of Animals with Tumors Zymbai Gland Carcinomas Nephrobla stomas Angiosarcomas Liver Other Sites Brain Neu robinHepatomas stomas VC, 10,000 ppm II VC, 6000 ppm in VC, 2500 ppm - IV VC, 500 ppm V VC, 250 ppm VI VC, 50 ppm VII No treatment Total 30 30 30 30 30' 30 40 220 1 4 4 10 8 11 23 61 1 (10) -- (3) -- (1) -- (2) _ 1 (5) 3 (3) ____ (4) ____ (2) ____ (1) 8 (4) 2 (3) 2 (4) 2 1 -- (2) 1 (1) 1 (2) -- 1 1 (16) 4 (13) 15 (11) 3 (5) -- (1) - 1 (1) -- (I) -- (2) -- 1 (2) 1 (2) -- (2) -- -- 1 (5) 2 (6) * In parentheses are recorded the number of tumors observed after 88 weeks among a comparable group of male Sprague-Dawley rats treated in the same way. Maltoni: Predictive Value of Bioassays 437 Table 5 Experiment BT5: Exposure of Breeders by Inhalation to VC in Air at 10,000 and 6000 ppm 4 Hr Daily for 1 Wlek (from 12th to 18th Days of pregnancy). Results After 115 Weeks Groups and Treatment I VC, 10,000 ppm, breeders II VC, 6000 ppm, breeders hi VC, 10,000 ppm, offspring IV VC, 6000 ppm, offspring Total Animals (Sprague-Dawley rats) Total Survivors 30 30 54 12 32 8 146 20 No. of Animals with Tumors Zymbal Gland Cardnomas 1 Nephrobla- stomas Angiosarcomas Liver Other Sites 1 -- --1 3 1--2 1 -- --2 5 1 --5 however, that tests of agents should follow a pattern that progresses in degree of precision and scrutiny so as to eventually filter out and expose the most dangerous compounds. Finally, 1 should mention that we must place emphasis on both scrutiny of compounds already produced and in widespread use and on compounds yet to be produced, before economic, social, and political interests become so en trenched that solution of these problems will be difficult indeed. Table 6 Experiment BT14: Exposure of Newborn Rats by Inhalation to VC in Air at 10,000 and 6000 ppm 4 Hr Daily, 5 Days Weekly, for 5 Weeks (FROM 1 DAY TO 5 WEEKS OF ,AGE) Results After 48 Weeks Groups and Treatment 1 VC, 10,000 ppm II VC, 6000 ppm Total Animals (Sprague-Dawley rats) S and c? No. of Animals with Tumors Zymbal Angiosarcomas SurviTotal vors Carci Nephrobla Other nomas stomas Liver Sites Hepa tomas 46 42 ~ 1-- 1 43 41 89 83 -- 1-- 2-- 2 3 EC 438 Annals New Y ork Academy of Sciences Experiment BT4: Table 7 Exposure by Inhalation to VC in Air at 10,000 , 6000, 2500, 500, 250 and 50. ppm for 4 hr Daily, 5 Days Weekly, for 30 Weeks. Results After at Weeks (end of the experiment) Groups and Treatment I VC, 10,000 ppm II VC, 6000 ppm III VC, 2500 ppm IV VC, 500 ppm V VC, 250 ppm VI VC, 50 ppm VII No treatment Total Animals (Swiss mice) 9 and <3 Total Survivors 60 -- 60 -- 60 -- 60 -- 60 -- 60 -- 150 -- 510 -- No. of Animals with Tumors Pulmonary Mammary Tumors Carcinomas * Liver Angiosar- comas Vascular Tumors of Other Types and/or Sites Epithelial Tumors of the Skin t Pregastric Papillomas 35 13 8 9 3 1 38 8 5 9 6 1 30 9 11 12 3 1 38 7 11 18 1 -- 33 11 11 22 2-- 2 12 1 13 -- -- 8 ____ -- 1---- 184 60 47 84 15 3 * Most included squamous metaplasia, t Some occurred in sebaceous glands. r, , Tahlf. 8 439 1939 Table 8 Experiment BT8: Exposure by Inhalation to VC in Air at 10,000, 6000, 2500, 500, 250 and 50 ppm 4 hr Daily, 5 Days Weekly, for 30 Weeks, Results After 76 Weeks Groups and Treatment I VC, 10,000 ppm No. of Animals with Tumors Animals (Golden hamsters) Liver Tumors o Angiosar- Angio- Hepa Total Survivors comas mas tomas Skin Trichoepi- Basaliomas mas Pregas- tric Papillo mas and Lympho Acantho mas * mas 35 1 6i 4 II VC, 6000 ppm III VC, 2500 ppm IV VC, 500 ppm 32 3 33 4 33 4 --2 --3 i 2--_ 22 I 3_ V VC, 250 ppm VI VC, 50 ppm VII No treatment Total 32 4 33 5 70 14 268 35 3 6 2 2 5 2 23 1 4 * Average latency times were 48 weeks in treated animals and 92 weeks in controls. 24 18 14 1 12 2 8 22 Others 2 (1 subcutaneous angioma) (1 adenocarci noma of gall bladder) (t subcutaneous angioma) (1 bronchial carcinoma) 4 2 3 hp rn$ Q. o' o < crs". O W VoJ) ' *S<J Vi 440 Annals New York Academy of Sciences Table 9 Experiment BT11: Exposure by Ingestion (stomach tube) to VC in Olive Oil at 50.00,-16.65, and 3.33 mg/kg Body Weight, Once Daily, 4-5 Days Weekly, for 52 Weeks. Results After 55 Week. Groups Treatment I VC, 50.00 mg/kg II VC, 16.65 mg/kg III VC, 3.33 mg/kg IV Control: olive oil Total Animals (Sprague-Dawley Total Survivors No. of Animals with Tumors Zymbal Gland Carci- Nephrobla nomas stomas Angiosarcomas Other Liver Sites 80 57 80 66 80 62 80 68 320 253 11 ---- 1-- -- -- ---- -- -- ---- -- 21 Table 10 Hepatomas Observed So Far on Rats and Hamsters Exposed by Inhalation to VC in Air, 4 hr Daily, 5 Days Weekly Animals Species, Strain, Age Sprague-Dawley rats 13 weeks old Sprague-Dawley rats, 1 day old Wistar rats, 11 weeks old Golden hamsters 11 weeks old Total No. 60 69 72 74 67 46 43 30 32 33 Treatment 30,000 ppm, 52 weeks 10,000 ppm, 52 weeks 6000 ppm, 52 weeks 2500 pprp, 52 weeks 500 ppm, 52 weeks 10,000 ppm, 5 weeks 6000 ppm, 5 weeks 6000 ppm, 52 weeks 6000 ppm, 30 weeks 2500 ppm, 30 weeks Hepatomas 1 1 1 2 3 1 2 1 1 1 14 M a lto n i: Predictive Value o f Bioassays EC- 194 Table 11 Tumors Presently Correlated to VC Exposure (by inhalation) on Experimental Rodents and Man Species Rat Mouse Hamster Man Angiosar comas of Liver Tumors of Brain ++ + + + (+) Tumors of Lung "f* (+) (+) Tumors Lympho mas and Leuke mias Angiosar comas and Angiomas of Other Nephro Sites blastomas Seba ceous Cuta neous Carci nomas (+) (+) H+ (+) ++ + Squa mous Tumors Mammary of Epi Carci dermis nomas + (+> ++ + Pregastric Papillomas and Hepato Acantho mas mas + (+> (+) + + + 442 Annals New Y o rk Academy o f Sciences 1942 Table 12 Experiments BO 12 and BT2001: Treatment by One Subcutaneous Injection OF 30 MG OF THE COMPOUND IN 1 CC OF H;0 Groups Treatment I, Chromite H. Neochromium HI, Chromium atlumen IV, Chromium yellow (lead V,- Chromium orange (lead chromate) VI, Molybdenum orange (lead chromate, sulfate, and molybdate) VII, Cadmium yellow (cadmium sulfide) VIII, Iron yellow (iron oxide) IX, Iron red (iron oxide) x, Control Animals (Sprague-Dawley rats) Total 40 40 40 40 40 Survivors ____ -- -- -- 40 40 -- 40 -- 40 -- 140 -- Tumors (sarcomas at the site of injection) No. of Animals with Tumors Average Latency Time (weeks) Histology Type -- -- -- rhabdomyosarcomas 9 78 and fibrosarcomas rhabdo myosarcom as 8 72 and fibrosarcomas rhabdomyosarcomas 26 40 and fibrosarcomas rhabdomyosarcomas 27 47 and fibrosarcomas 36 32 rhabdomyosa rcotnas and fibrosarcomas rhabdomyosarcomas 16 86 and fibrosarcomas 1 77 rh a bdomyosarco mas and fibrosarcomas rhabdo myosarcom as and fibrosarcomas ---- '' Length of Experiments (weeks) 133 149 137 150 150 117 143 125 134 127 Maltoni: Predictive Value of Bioassays 443 Experiment B08: Table 13 Treatment by One Endoperitoneal Injection of 25 mo of Crocidolite in 1 cc of Saline Groups and Treatment I, Crocidolite II, Saline (control) Animals rats) (Sprague-Dawley Sex No. $ 50 50 9 45 J 15 Cor rected No.* 49 48 45 15 No. 34 31 -- . --- Animals with Peritoneal Mesotheliomas i Average Latency Time % (weeks) Animals with Metastases 69.3 64.5 -- -- 73 65 -- -- 13 38.2 13 41.9 -- -- -- --- * Animals alive when the first peritoneal tumor arose. References 1. Lacassagne, A. 1938. Apparition d'adenocarcinomes mammaires chez des souris males traitees par une substance oestrogene synthetique. C. R. Soc. Biol. 129: 641. 2. Lacassagne, A. 1950. Les Cancers Produit par des Substances Chimiques Endogenes. Herman. Paris, France. 3. Van Duuren, B. L., A. Sjvak, B. M. Goldschmidt, C. Katz & S. Melchionne. 1969. Carcinogenicity of halo-ethers. J. Nat. Cancer Inst. 43: 481-486. 4. Laskin, S., M. Kuschner, R. T. Drew, V. P. Cappiello & N. Nelson. 1971. Tumors of the respiratory tract induced by inhalation of bis(chloromethyl) ether. Arch. Environ. Health 23: 135-136. 5. Maltoni, C. (Ed.) 1974. Occupational carcinogenesis. In Advances in Tu mour Prevention, Detection and Characterization. Vol. II: 19-26. Excerpta Medica. Amsterdam, The Netherlands. 6. Maltoni,. C. & G. Lefemine. 1974a. Carcinogenicity bio-assays of vinyl chlo ride. I. Research plan and early results. Environ. Res. 7: 387-405. 7. Maltoni, C. & G. Lefemine. 1974b. Le potenzialita dei saggi sperimentali nella prcdizione dei rischi oncogeni ambientali. Un esempio: il cloruro di vinile. Accademia Nazionale dei Lincei, Rendiconti della Classe di Scienze Fisiche, Matematiche e Natural!, Vol. LVI, Ser, VIII, Fasc. 3. 8. Maltoni, C. & G. Lefemine. 1975. Carcinogenicity bio-assays of vinyl chlo ride: current results. Ann. N, Y. Acad, Sci. 246: 195-218. 9. Maltoni, C., G. Lefemine, P. Chieco & D. Carretti. 1974. La cancerogenesi ambientale e professional: nuove prospettive alia luce della cancerogenesi da cloruro di vinile. Gli Osped, Vita 1: 5-6, 4-66. 10. Maltoni, C,, A. Ciliberti, L. Gianni & P. Chieco. 1975. Insorgenza di angiosarcomi in ratti, in seguito a somministrazione per via orale di cloruro di vinile. Gli Osped. Vita 2(1): 65-66. 11. Viola, P. L. 1970. Cancerogenic effect of vinyl chloride. In Xth International Cancer Congress, Houston, Vol. 29. 12. Viola, P. L-, A. Bigotti & A. Caputo. 1971, Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res, 31: 516-519. 13. Selikoff, I, J. 1975. Personal communication. 14. Popper, H. 1975. Personal communication. EC- 1943